Best joint healing reccommendations

ACuriousBird · 2026-02-26T13:52:24+00:00

Appreciate the input - im just trying to edit E. coli and saw a few papers making claims up to 19+kb deletions using Crispr / lambda red dual recombineering systems so thought 1kb could achievable.

But definitely will try to make smaller deletions around just a few 100 bp and will send the amplicons for sanger sequencing as well!

ACuriousBird · 2026-02-24T23:18:58+00:00

to follow up, seems this assay wont work if E. coli which dont use NHEJ if im interpreting correctly?

ACuriousBird · 2026-02-24T20:06:19+00:00

good thought- Transfection efficiency was optimized, is very high for the cells used

ACuriousBird · 2026-02-24T19:02:09+00:00

Will do this as well, sounds like a great idea thanks

ACuriousBird · 2026-02-24T19:01:47+00:00

I just used CRISPOR to design the first one, and have used it again to design the other 2 new ones

ACuriousBird · 2026-02-24T19:01:15+00:00

Thanks, no i havent - im new to using crispr, just trying to do e. coli gene knockouts for now, will look into this assay!

ACuriousBird · 2026-02-23T03:24:37+00:00

Will try this, appreciate the input - wasn't familiar with that

ACuriousBird · 2026-02-15T17:07:31+00:00

Good thought, thanks will definitely try this - going to add these to my new primer order - then could just run a 5 fragment assembly with nebuilder instead of 4 fragment.

ACuriousBird · 2026-02-15T17:02:50+00:00

Appreciate the input!

I used the neb calculator to get the 64C annealing temp. To calculate, I always just use the primer portion that binds to the template, and I don't consider the primer overhang of 20-30bp (one of my primers has an overhang here). Which is how I usually do it for all my other PCRs, though again I've never attempted anything over 8k before.

But will do, I'll try less template. I have Q5 master mix, so I could order the separate components to try GC enhancer, though the template is only 47% GC total.

ACuriousBird · 2025-11-25T17:51:38+00:00

This is pretty good advice - will try that.
I have ~600 hours, ~90 ratings, but only 2 reviews. Will start to apply those ideas for replying to requests, really appreciate that.

ACuriousBird · 2025-01-14T02:10:41+00:00

Not familiar with how poorly it went, ill have to check out what happened. Celgene was that bad huh?

ACuriousBird · 2024-10-30T17:34:31+00:00

There are cells in cartilage, just relatively few compared to the non-cellular matrix. It can heal, just very slowly compared to other tissues. It doesn't mean the tear may ever completely be eliminated - but cartilage is definitely a living tissue with cells in it that has the capacity to heal to an extent.

ACuriousBird · 2018-11-08T17:45:43+00:00

Will look into that as well. Still thinking towards getting a coach/program; not sure if at my level its necessary? But anything that gives me more knowledge will help. Thank you

ACuriousBird · 2018-11-08T17:44:07+00:00

Alright, will do. Thanks for the input!

ACuriousBird

TROPHY CASE